The culture process for mammalian cells, animal cells, insect cells, bacteria, yeast and molds has one major rate-limiting step, oxygen mass transfer. Oxygen metabolism is essential for metabolic function. In mammalian and animal cell culture, oxygen flux is especially important during the early stages of rapid cell division. Oxygen utilization per cell is greatest when cells are suspended; requirements decrease as the cells aggregate and differentiate. However, during the later phases of cell culture, as the number of cells per unit volume increases, the bulk oxygen mass transfer requirements increase. Traditionally, increased requirements are accommodated by mechanical stirring methods. The present invention integrates several features needed for the engineering of autologous tissue transplants. Incorporated into the invention are; automated cell culture media repletion, the capability to grow cells in monolayer, a turbulent hydrodynamic cell culture environment suited to the growth of three-dimensional tissue constructs, a unique configuration that allows both monolayer and 3-dimensional culture in a variety of combinations, a gas head space for direct oxygenation of the cell culture media, an internal sensor for aseptically monitoring media changes, the capability to aseptically regulate both media repletion and add turbulence for improved mass transfer, and all features are included in a very low cost disposable two chamber tissue culture roller bottle. It will be appreciated by those of ordinary skill in the art, that the invention eliminates a large portion of the cell culture labor costs. The instant invention; 1) establishes near physiological control for improved engineering of tissue constructs, 2) enhances mass transfer through hydrodynamic shear forces generated at the wall and through the turbulent interface with the first chamber, 3) eliminates cellular contact with a potentially toxic silicone membrane oxygenator because oxygenation is predominately through the air fluid interface, 4) increases the concentration of de novo growth factors because the media is not replaced as often, and 5) greatly reduces the risk of contamination.
Culturing tissue for transplantation requires that several conditions be met before the tissue receives Food and Drug Administration (FDA) approval. Those FDA conditions include; functionality that ameliorates the disease; consistent and reproducible growth of tissue constructs; and proven sterility. To achieve in vivo functionality, engineered tissue constructs must be three-dimensional. To be reproducible, the cell culture environment should be regulated to match human physiology, a feature of the invention. Data from the aseptic monitoring of the growing construct can be used to validate sterility and establish specifications.
Transplantable tissue has three key features; 1) an extracellular matrix for mechanical stability and scaffolding; 2) cell-to-cell contact to maintain viability and function; and 3) a three-dimensional shape to segregate cell subpopulations for growth and proliferation. Standard tissue culture approaches (e.g.; t-flasks, petri dishes, microgravity culture vessels, roller bottles and stirred roller bottles) have consistently failed to yield transplantable tissue that directly supplants organ function. Failure is often related to the loss of multi-dimensional cell-to-cell contacts and the overgrowth of unwanted cell subpopulation. In roller bottles and stirred reactors, cells are prevented from sustained three-dimensional growth. Shear preselects for only those cell subpopulations that are robust to its damage. The present invention controls the addition of turbulence and concomitant shear, and localizes shear to specific areas. There are other selection pressures that impede normal growth, besides those intrinsic to standard cell culture roller bottles. Specifically, those technical operations associated with feeding the culture. Standard cell culture practice replaces cell culture media at regular intervals or when the media changes color. Replacing the media at regular intervals dilutes growth factors that are vital to cell growth and proliferation, and typically exposes cells to abnormally high oxygen concentrations. Abnormally high oxygen exposure occurs when spent cell media is replaced with fresh media. Fresh media equilibrated to room air has a dissolved oxygen concentration of approximately 159 mm Hg, often more than double the normal concentration found in the average mammalian interstitial space (normal 40-80 mm Hg). High levels of oxygen are known to be toxic to highly differentiated cells. Therefore, the practice of replacing conditioned media with fresh media equilibrated to ambient air, preselects for cells that are less sensitive to oxygen toxicity (e.g.; fibroblasts). Changing media based on the pH indicator dye, phenol red, imposes another common environmental selection pressure. Human physiology maintains the vascular and interstitial pH between 7.30-7.40. Deviation beyond physiological limits often results in mental confusion and organ dysfunction. Yet, most cell culture facilities routinely maintain the pH within 7.00 to 7.45. If patients were repeatedly exposed to standard cell culture pH extremes, they would eventually succumb to multi-system organ failure.
There are several deficiencies associated with continuous perfusion of cell cultures. Continuous perfusion consumes large amounts of cell culture media, serum, and growth factor supplements. The expenses associated with perfusion reactors preclude their general use in research, and perfusion often yields tissue constructs that are delicate and of poor functionality. There are several additional drawbacks to profusion culture; cell populations are first manually expanded as monolayers of cells, yielding an atypical architecture; subpopulations that are robust and grow rapidly, overgrowing the other populations; and the cells must be anchorage dependant to keep from being washed away in the perfusion stream.
A three-dimensional architecture is required for tissue constructs to be functional. As aggregates of cells organize, they exhibit the architecture of nascent tissue. To create a tissue construct, dissimilar cell types must aggregate, free from the selection pressures that force cells to spread and grow along a flat surface. Hydrogels and media viscosity enhancers permit cell mixtures to remain in contact with other cells, thereby allowing three-dimensional tissue growth. However, it has been observed that the sizes of the constructs were small, and latter deduced that they were mass transfer limited.
Roller bottles are ubiquitous to cell culture facilities, and have been in use for decades. Their strengths are; they are disposable and offer a large surface for cell attachment. Their weakness is a consequence of the large uncontrolled hydrodynamic shear associated with a gas headspace and the abundance of turbulent eddies. The high shear environment inherent in roller bottles precludes their use for growing tissue for transplantation. Only those cell types that are not damaged by the shear and adhered to the wall, can be maintained in culture for the extended periods.
Horizontal rotation for suspension i.e., clinostatic rotation, began in the 1700s. The physics for growing cells in a free fall or in a microgravity like environment, was first characterized in detail by the Argonne National Laboratory (R. R. Dedolph and M. H. Dipert, 1971). Those principles were applied in the NASA microgravity culture vessels and cell culture methods, and the vessel and culture methods were first disclosed by M. Lewis et. al., May 1987. Later, the tubular silicone oxygenator in the culture vessel of M. Lewis et. al., 1987, was replaced by a membrane silicone oxygenator, and the culture vessel patented by NASA--U.S. Pat. No. 5,026,650 issued June 1991. Culture systems developed by NASA strive to achieve a quiescent microgravity like environment, having origins in the NASA space program. Microgravity culture conditions are created by low shear and essentially no relative motion of the culture environment with respect to the walls of the culture vessel (U.S. Pat. No. 5,496,722; claim 12). Essentially no relative motion differences between the culture media and the vessel wall, is often referred to as solid body rotation. The forces maintaining solid body rotation are associated with the 1:1 viscous coupling between the vessel wall and the cell culture media contained in the vessel (G. F. Spaulding, 1994). However, microgravity is known to cause aberrancy. There are over 100 scientific articles and abstracts demonstrating or suggesting microgravity induced aberrancy in cell growth, cell differentiation, cell movement, cell size and shape, cell physiology, apoptosis, abnormal cell cycle, abnormal phenotypes, or general human physiology, occurring in rotating culture systems or in actual microgravity. The normal mammalian hydrodynamic milieu differs from a quiescent microgravity like environment. In utero, cells are constantly moved, pulled or forced to settle in different directions depending on the position of the organism, i.e.; standing, sitting or lying down. Movements from walking, running, and breathing agitate the cells; thereby adding hydrodynamic shear. An intrauterine environment is characterized by cells developing in amniotic fluid under constant hydrodynamic shear, resulting in high mass transfer. The present invention more closely emulates the intrauterine hydrodynamic and gas mass transfer environment where positional changes, breathing, movement, and hemodynamic pulsations continuously disrupt boundary layers. At the same time, the maternal physiology closely regulates the intrauterine environment; facilitating normal growth of organs. Major changes in maternal physiology would elicit great concern for abnormal fetal organ development. Yet, the standard of tissue culture practice for those of ordinary skill in the art, is to allow the cell culture pH and oxygen to fluctuate beyond in vivo limits.
The invention, herein disclosed, integrates a novel hydrodynamic cell suspension environment, with regulation of media repletion and pulsatile mass transfer or diffusive, to achieve three-dimensional tissue constructs suitable for transplantation. An automated tissue culture system that facilitates three-dimensional growth, and is disposable, fulfills a need for technology to engineer autologous tissue constructs, as well as grow cells for basic research.